Materials. Perfluoro-15-crown-5 ether (PFCE, C10F20O5) was purchased from Exfluor Research Corporation (TX, USA). High-purity lecithin, cholesterol and 2-dipalmitoyl-snglycero-3-phosphoethanolamine-N- (lissaminerhodamine B sulfonyl) (16:0 LissRhod PE) were purchased from Avanti Polar Lipids, Inc. (AL, USA). AZD9291 was purchased from Medchem Express (NJ, USA). Glycerin was purchased from Aldrich Chemical Co. (WI, USA). Argon was used for the storage of materials. The Spectra/Por membrane (Cellulose MWCO: 20 kDa) used for dialysis was purchased from Spectrum Medical Industries, Inc. (CA, USA). Zoletil™ 50 was obtained from Virbac (Nice, France). All chemicals used in this work were of analytical grade and used as received.
Preparation of AZD9291-PFCE nanoparticles. AZD9291-PFCE NPs were synthesized by microfluidization method according to the procedure reported previously.30–32 Briefly, the nanoparticulate emulsions were comprised of 20% (v/v) PFCE, 3% (w/v) of a surfactant commixture, 2.5% (w/v) glycerin and water for the balance. The surfactant co-mixture of NPs included 73 mol% lecithin, 14.9 mol% cholesterol, 12 mol% AZD9291, 0.1 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissaminerhodamine B sulfonyl) (ammonium salt) (16:0 LissRhod PE), and 0.05 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-((folate)amino)hexanoyl) (sodium salt) (16:0 Folate Cap PE) on the outer shell to endow an FR targeting. The surfactant components were combined with PFCE, water and glycerin, then homogenized at 20,000 psi for 4 min with a microfluidizer M110P (Microfluidics, Newton, MA). Procedures were made following dialysis (20 kDa MWCO cellulosic membrane) of nanoparticle suspensions into deionized water. The resulting emulsions were conserved under argon in aseptic sealed vials until use.
Characterization of nanoparticles. Transmission electron microscopy (TEM) images and elemental mapping analysis were characterized using a FEI Talos F200X instrument equipped with 4 Super-X SDDs. The size distribution and zeta potential of NPs were measured using a Zetasizer Nano ZS90 (Malvern Instruments). The absorption spectra were determined by Perkin-Elmer Lambda 35 UV/Vis spectrophotometer (Perkin-Elmer, USA). To evaluate the imaging properties of AZD9291-PFCE NPs, the phantoms with various 19F concentrations were investigated by 19F MRI and fluorescence imaging. The AZD9291 concentration in AZD9291-PFCE NPs was examined by high performance liquid chromatography (HPLC). The encapsulation efficiency (EE) of AZD9291-PFCE NPs was calculated as follows:
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SQwX0unLp4spbuOOONUuHhMqiNMONoxpfDX4z/s7/Cj4KeI/h1pfgH9ou58GeLrjU7nWNN1j4Q/EXWVv31Jne+3veadLJtneWV3QMFLSyNjLsT9m0UAfnuPCv7Jb+DPF2gT/Dv9qa903x74ctvCXiAXvgH4q3U+qaVbzTTQWsk0lq0u2MzzKpDBhFI0WfK+Sut+PvxG/Zv/AGn7bQo/G/gT9pfV/wDhHdMvdFtJIvhb8S7KWaxvUhS7tbiS3sY2uoZhbQF0uDIrNEjEFhmvtuvG/iz+2p4X+C3x5ufBmuyPAumeAdR+IOoSQ6dqt3drY2l1b27tbxQWUkFyF85jIiXH2mMmDFu6TeYgB8KeOfBXhe7+P+meJPBfj79qLwL4U0600PTl0my+CvxW/tGO10tEjjh+0xPFaXRkRSryalY3spEjguRtC/Qvxs+OP7Pv7QnirSNb8T+Cv2lZdV0SF7WC4034T/EjSWuLZ2V3tbkWmnxC6tmZFLQTiSI45Q5Ndp4p/wCCqvwoT4M+M/F3hzW5dUj8F2mlX10NR0LW9PtmttTkZLG8Dpp80slnMY5dt1BDND+6fLAKxFD41f8ABWLwD8IrT42QS6T42ttR+CEIfVbzV/B2vQeH5maG2kVv7RtNPu9sQ+1R7mETuEWSZY3gTzSAea+N9X/Zi+InjvXfEuqeBv2pn1jxL4h0jxVqM1v8O/ilaJNqOlRtHYTLHDaJHGsSsP3aKsbtHEzo7RRlW3N7+yxeaL450yT4a/tIHSfiK1zJrOnf8Ks+JYsUkuLkXVxLZ24sfKsJJLlVneSzWF2lVXJLAGvp/W/2w/AGnfH60+FP9vTReO9Ukks7OE6LfTWAvFsWvzaveLGLVbkWa/afszTrMYcOF2kNXI/Bz9s+PR/2B9M+MPxYuLaxW0s5ZteufD2hX9zBGY7p7cyQ2cX2m5CEqCRmQqCSWwCaAPkr41eBfgv40svDWneDf+GnPBulQ+PrPx14pNx8JfitqWp+IZoLJrEiDUAsNzYXDwMN1zE7Mzxxs6yYcP2Wr+Hv2SNc0fw9p8/wu/aSWw8NxSQQ20Pwx+J0EWoJLefbpRqCpZAaj5l2TO5vfOLysztlmJP1FJ+3t8KU8VPoaeJbi41iLxdaeBJLO30W/nmj1m5tRdxWzKkJIUQbneY/uYhDMJHQwyhOH+If/BVv4TeH/gP8RPG2ha6dUg+H2jrrUzX+ia3ZWN7aySmCK9hmi0+eW508yqQ17ZQXUSKrOSVUmgDy3Wde/Zh1/wCOMfxGufh9+0z/AMJXD4ig8WxTRfDH4mw2kWqxWhshdrZpZC1WRrX9zJiLEyBVkDhRjmrvwF+yHfeH4tMm+Gv7UctrE2u7i3gD4qma4j1uRJdVgml+y+ZNBcyosrwyM0Xm5kChyWP078SP+Cj3wi+FHx1t/htq+t+IW8XXOr2WgC307whrOp2sOoXcH2m3tJbu2tZLaKZ7f99sklVhEDIwCAtXn37LP/BT7QvGfwynsvH97JP8TdBsfEWpajZeHvB2rw2esW2j6nLY3Emmq4nW5kUC3L28NxPLG067gARQBx8/i/8AZlvbid7r4fftKX63vhKHwPfQ3vws+Jd1b6ppUMUsUUd1FJYtHczIk0wFzMr3A81z5uWJMN94k/Zo1X4X2vhK88F/tVXthYa1H4htL+5+HvxSm1yzv44zDHcRaq9ob+NkiZol2TgLG7IAEYqfoj4X/t1/D/4nfspJ8Z3l8R+FPA/2CPUJ5vFHh+90a5gjeOORcQzxK0wPmoqPB5scrHETycV5N+0f/wAFVfDmifAm21r4aPrOpa/qXi+z8FSRah8PfEN5deGbueMXBlvdGit4tROLch44sQmYyRhXwc0Ac9p3xC/Zr0n9mzxX8Ibf4fftHxfDzxu+pvrOlD4T/EjN1/aUsk18qz/YPPiSZ5ZSyRSKv71wAAxBwviMn7LPxW1DxFdaz4C/ane48V2ejafqj2fw++KdgbiHSJDLpqjyLRPLMErPIrJtYu7MxZmJPsX7dP7ch/Yy/Zk1OW91TTJ/jBH4PvdcsLCw8Katr1jPcWcCtPcTW1lunt9P85lQzzyxxxiVd0nBzcsv+CmXw08Jv4N0Hxnq+paZ448TaToF81hZ+FtWurcvq8iW1qyTRW8kXkvduIN5kKxyPGkjKzruAPP/AI1/HH4AftCeNdE8ReKPB/7Tc+seHdM1TR9OuLD4WfErS/s9tqVv9mvF22thErO8eNsjAvEyI8bI6qw8713wJ+yJ4i0LTtOn+G37Ukdppfh/TvC8ItfAHxVtXfT9OuGudPjleK1VpntZmZoZpC0sWcI6jivqLQP+Cinwe8R2NpdReK7i1tb3wVf/ABDjnv8ARNQsIl0OwmSC8unaeBBG0MksYeFyJlDgmPac1o/s0ftyfDr9rrXvEmmeB7zxTcX3hEWv9rQ6v4P1jQTa/aYhNAM39rAHLxFZAE3HY6N911JAPmyfV/2Yrj4gv4oPgX9qRNak8bJ8RXlj+HXxRjik11LdbVbpoVsxEV+zosJhKeSYhsMZXisS6i/Z28G+F/GP/CvPBnx+8J+KPEeg6noWm3+o/BX4ka5p3htL/wCa4+x6dNaLDbRvIqO8VqbcSGNdx4FfoVRQB+cPwT8Ffs1fDv8AZwk8B+JfCP7SHjK81nRtK0nX9ZufhP8AFE3N6NOdZbX7JJcW9xc6dDFOvnRQ21wqwvymCM11egXv7L/hnVdGvbXwJ+1KbnQPF7ePLF7j4efFK58vW2tmtXvGElowkZ4XkV0fcj+dKWUmRy33nRQB8NfF7xb+zN8drrx9J4n8A/tM3ifFDTI9I8UW1v8ADD4mWNpq1uiog3QQWKRJIY40iaZFWVo18tnKErV3Ufih+zpqXxa1nxx/whX7Ttr4l8RWYs9Tnsvhh8TLOG/K2v2RLiS3isVha7S3xGl2U+0Rqq7ZF2jH2xRQB8EaKf2WPDfh3wTpWnfD79p+wsPh7pcehaPHbfDj4oxE6bHJ5kdhdMtmGvrRG+7b3ZmiUfKFC8V3n7OEX7P2q/taHxR4S8E/HLT/AB94hk1GaO88SeBvHenaJYyXWLi9kiGp2yaZYPOYQXZBEZXCrlmcKfruigCG/SeSxmW1kihuWjYQySxmREfHyllDKWAOMgMM+o6180fsxftveJP2ifj1deBJNH0fQm8FR3D63rEqTvYeOVj/AHPneHGJXzraGaSL7XO5k+yzbbXZKZluk+ldU0u21zTLiyvbeC8s7yJoLi3njEkU8bAqyMpyGUgkEHgg1l/8Kz8N7fDg/wCEf0THg9/M0EfYYv8AiSN9nktd1r8v7g/Z5ZYcx7f3cjp91iCAfKv/AAX88A2Hjz/gkJ8c/t8mrR/2L4ZutVtxY6rdWAkmijbYJhBIgnh+Ykwzb4mIUshKqRJ+1FoGn/Hr9sD9nX4Q+MtHtPEHw11vwx4h8VatoupQC40vXrzT00yC0guoHBjuI4v7QmnEcgZfNihk27okYe2ftrfsm6f+3D+zj4h+GGteKfFfhTQPFcJs9Wn8PGyW7vLVlZZLfdd21wiI+RlkRZBtG115zn/EH9jSD4jeA/A1nc+P/Hll4z+HUpm0Tx1ZjS49ehd4Wgm3xmyOnyJNC5R43szHwjhFkjjkUA+HP2fLtP2kPjp8PPgF45sh4t+Enhjxd8UYY9K1ofb7DWodB1e1s9KsbuOQGO6itIr+TZHLvAezt3I3wqw4n4gfs1/E39rn4FXWheHvDF38UvDfwy8SfFD4ceG9OuNVtIX8MagNQFt4e1wzXUsfGlpFcW6zQGS7hVh5SOxfH6Dap/wT28JRfCrwP4e8Na94v8E6x8Or251PQ/Fek3NtNrcN3eecdQnma7gnt7lrxrid5xPA6NJL5gVZEjdOq8NfsneHPBf7Olr8MNHvdc07w8jM19Mlwkl9rPnXLXN99pmkRizXskk5nkQJITcysjxuVZQD5j+Jv7enhj4Ha54M8QfEKx8VeOdP8L+GbvxDptzoVvbXcUGmWC29pqvjO4Es0Ra2zerHbi2E0zwSzSxJKs+I/ue1u4r61jngkSaGZQ8ciMGV1IyCCOCCO9eC/te/8E5fA37ZVzp0mt6j4n8Nm20O98JagPD1xBajXNAvXt5LzSLjzIZCttM1pBloPKnTYRHNGGbPvVraxWNrHBBGkMMKhI40UKqKBgAAcAAdqAJKKKKACiiigAooooAKKKKACiiigAooooAKKKKACiiigAooooAKKKKACvGvjV+xZo3xu+MVz42u/FHi7SNUm8Aax8PIodOay+z2lpqctvLcXaCa2kY3Qe0t9hdmiHlcxNubd7LRQB8j6l/wSB8Mat4A8R+Gp/ij8Wm0zxN4E8O/Du4XzdFDQ6bok7zWjRn+zsCZjPdiRmDKRdy7VQrEY+u+Pn/BN/QP2g9P+MOnah47+Imj6L8cNIj0rxHpmmS6asCstvFatdQNNZySJM9tCkLBnaIAlljWTDj6KooA+WNc/wCCTPgzW/2wLD43/wDCZePIPHOk66NesLgR6PMbItp0unXFnHPLYPdCynhldntvO8tZMPEIjXpXwf8A2NtB+F37JU/wa1HW/Enjnwxe2F/pd5c+IGtPt15bXrSmaJzawQRYxM6jEYOMZJPNeu0UAeCfBP8A4J0eAvgZ8XND8c2Nz4k1bxLovhuPw811ql4k39pSrNczPqtwqxqr6jI17eB7gBcrdSqFCtivIfAf/BBP4J/DD4GfEjwB4dl1vQ9M+JXh9vCl1f6bpeg2Wq6dpTMGktY7qLTlkuN+AGlvjdTcAiQN81fbNFAHwd4t/wCCdPxf1H/gpdJ8YNN1fwBFoz+IdNv49UvY9OvtVt9LhtLK1vLFIJ9Clu4XuEtpvntNYtoUa48z7MWEon9U+EX/AAS68P8Awo8VeCNWl+I/xP8AFE/gWx8Tafapq8mkhL6PxBcx3eoef9msIWJ8+KJ4/LKBNgGCpIP07RQB4ZZ/sFeH5v2LF+BmteKfG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In vitro drug release. The release property of AZD9291-PFCE NPs was determined using a dialysis method at different pH values. Briefly, 1 mL of AZD9291-PFCE NPs was sealed in a dialysis bag (MWCO 3000 Da) and immersed into 400 mL of phosphate buffered saline (PBS) solution (pH 5.0, 6.5 and 7.4) at 37°C with continuous shaking. At the time points of 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h, 1 mL of release medium was collected and replaced by 1 mL of fresh buffer to maintain the sink condition. To investigate the LIFU-triggered drug release profile of AZD9291-PFCE NPs, 1 mL of AZD9291-PFCE NPs were injected into dialysis bag with LIFU irradiation and dialyzed against 400 mL of PBS (pH 7.4) at 37°C in a shaking water bath. An acoustic frequency of 1.1 MHz, an acoustic intensity of 55.8 mW/cm2, a 50% duty cycle, pulse wave mode, a power of 3 W and a duration of 10 min were set as LIFU parameters for sonication. The next steps were similar to without LIFU group. The AZD9291 concentration in the collected buffer was measured by HPLC.
Cell culture and animal model. Human non-small cell lung cancer (NSCLC) cell lines, H1975 (EGFR L858R/T790M positive) and H520 (EGFR negative) were purchased from the American Type Culture Collection (ATCC). All cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL). Cells were maintained in a humidified incubator with 5% CO2 at 37°C. Female BALB/c nude mice (5–6 weeks) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal studies were approved by the Institutional Animal Care and Use Committee of the Harbin Medical University and in accordance with all related ethical guidelines. The female BALB/c nude mice were inoculated subcutaneously into the right thigh with 200 µL of cell suspension containing 1×107 H1975 cells. The tumors were allowed to reach 100 ~ 200 mm3 before experimentation. The tumor volumes were calculated by the following equation: volume = (length×width2)/2. Relative tumor volumes were calculated as V/V0 (V0 was the tumor volume when the treatment was initiated).
Cellular uptake analysis. H1975 cells (105 cells/mL) were seeded in 6-well plates and allowed to grow until 80% confluence. Then PFCE NPs or AZD9291-PFCE NPs labeled with rhodamine B were added at a final concentration of 3 mg/mL of PFCE. LIFU (55.8 mW/cm2, 50% duty cycle, pulse wave mode, 3 W, 10 min) was performed 1 h after incubation, respectively. After incubated for 4 h at 37℃, the cells were washed with phosphate-buffered saline (PBS) twice and fixed with 4% paraformaldehyde for 10 min. The cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Cell images were observed using a confocal laser scanning microscope (CLSM, Nikon, Japan).
Penetration in solid tumor spheroid. The three-dimensional (3D) tumor spheroids were established according to the reported protocol.39 Briefly, H1975 cells (5×104cells/mL) were seeded into 96-well plates pre-coated with 1.7% low melting point agarose and incubated for a few days. When the diameter of tumor spheroids reached to about 400 µm, rhodamine-labeled PFCE NPs or AZD9291-PFCE NPs were added into the spheres with or without LIFU irradiation (55.8 mW/cm2, 50% duty cycle, pulse wave mode, 3 W, 10 min). After incubation for 4 h at 37℃, the tumor spheroids were washed by PBS and fixed with 4% paraformaldehyde for 15 min, respectively. The Z-stack scanning for the spheroids was performed at 10 µm intervals by CLSM.
Cell viability assay. The cell viabilities were evaluated by MTT assay. Briefly, H1975 and H520 cells were seeded in 96-well plates at the density of 1×104 cells/well and incubated for 24 h. The experiment is divided into two parts. The first part was used to evaluate the cell viability of the H1975 and H520 cells treated with PFCE NPs, AZD9291 and AZD9291-PFCE NPs at different concentrations for 24 h and 48 h. The second part was used to assess the cytotoxicity of the H1975 cells incubated with free AZD9291 solution and various formulations of NPs with or without LIFU irradiation for 24 h. Part II included 8 groups: Control, PFCE NPs, AZD9291, AZD9291-PFCE NPs, Control + LIFU, PFCE NPs + LIFU, AZD9291 + LIFU and AZD9291-PFCE NPs + LIFU (n = 3). The groups of free AZD9291 and AZD9291-PFCE NPs had the same concentration of AZD9291 (0.1µM). LIFU was carried out after incubation for 1 h with different treatments (1.1 MHz, 55.8 mW/cm2, 50% duty cycle, pulse wave mode, 3 W, 15 min). After incubation for 24 h and 48 h, 20 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solutions (5 mg/mL) were added to each well and incubated for 4 h. After replacing the culture medium with 150 µL dimethyl sulfoxide (DMSO), the optical density (OD) values were measured at 490 nm using a Synergy HTX microplate reader (Bio Tek, USA). All experiments were performed at least three times independently. The IC50 of 24 h and 48 h in different groups was calculated by GraphPad Prism 8.0 software.
Cell apoptosis assay. H1975 cells were seeded in 6-well plates at a density of 5×105 cells/mL and cultivated in a humidified atmosphere at 37°C with 5% CO2 for 24 h. The grouping method of cellular apoptosis analysis was in accordance with the part II of cell viability assay. LIFU was applied at 1 h after different treatments with the same parameters described above. After 24 h, the cells were harvested, centrifuged at 1000g for 5 min, washed with cold PBS twice and resuspended in binding buffer. Afterwards, 5 µL of Annexin V-FITC and 5 µL of PI were added and incubated with the cells for 15 min in the dark at room temperature. The stained cells were measured using MoFlo XDP flow cytometer (Beckman Coulter, Fullerton, CA, USA). The results were analyzed using Summit 5.2 analysis software (Beckman Coulter).
The apoptotic cells were also detected by TdT-mediated dUTP nick end labeling (TUNEL, Roche, Switzerland) staining. Briefly, cells were cultured in 6-well plates before various treatment, then fixed with 4% paraformaldehyde for 20 min, and incubated with the TUNEL reaction mixture for 1 h at 37°C. The number of TUNEL-positive cells was observed by CLSM.
In vivo MRI-guided LIFU. Mice were anesthetized with 2% isoflurane and injected with PFCE NPs and AZD9291-PFCE NPs via the tail vein (300 µL, n = 3). The MRI-guided LIFU system is illustrated in Figure S8, Supporting Information. Mice were imaged with a 9.4 T MR scanner (BioSpec 94/20 USR; Bruker Biospin, Germany) using a 1H/19F double tune volume coil and their respiration rate was monitored during MR scanning. Proton anatomical images were acquired with an axial T1-weighted rapid acquisition with relaxation enhancement (RARE) sequence and the following parameters: repetition time (TR)/echo time (TE) = 820/12 msec, number of averages (NA) = 8, RARE factor = 8, matrix = 256 × 256, slice thickness (ST) = 1mm, and field of view (FOV) = 38.4 × 38.4 mm2. 19F MRI was implemented with a RARE sequence (TR/TE = 2000/100 msec, NA = 128, RARE factor = 32, matrix = 64 × 64, ST = 3 mm, FOV = 38.4 × 38.4 mm2). A reference phantom with 44.38 mg/mL of PFC emulsion in agarose gel was put beside the tumor for signal quantification. The 19F image were displayed by pseudo-color processing and overlaid on corresponding 1H anatomic images. 19F signal intensity was measured by the ParaVision 6.0.1 software and the fluorine concentration of tumors was calculated according to the determined concentration of the reference tube. Four hours after injection of NPs, LIFU (1.1 MHz, 55.8 mW/cm2, 50% duty cycle, pulse wave mode, 3 W, 20 min) was applied to trigger the location of the tumor and MRI was implemented synchronously. The permeability of PFCE NPs and AZD9291-PFCE NPs with LIFU irradiation was visualized and quantified by 19F MRI.
Biodistribution studies. 48 hours after administration of rhodamine-labeled PFCE NPs and AZD9291-PFCE NPs with or without LIFU irradiation (1.1 MHz, 55.8 mW/cm2, 50% duty cycle, pulse wave mode, 3 W, 20 min), the nude mice were executed and tumors, brains, lungs, hearts, livers, spleens, kidneys and intestines were harvested and observed using an IVIS spectrum imaging system (PerkinElmer, Waltham, MA, USA) with a DsRed filter (excitation passband: 500–550 nm; emission passband: 575–650 nm). Meanwhile, the removed tumor tissues and major organs were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned at 5 µm. The cell nuclei were stained with DAPI and then washed three times with PBS. The fluorescence intensity was detected to evaluate the distribution of NPs by CLSM. To further investigate the accumulated concentrations of AZD9291 in tissues, the H1975 tumor-bearing mice were treated with free AZD9291 via oral gavage and AZD9291-PFCE NPs by intravenous injection at a dose of AZD9291 (5 mg/kg), respectively. For the LIFU groups, tumors were triggered by LIFU with the parameters as described above. The mice were sacrificed at 48 h post treatment. The tumor tissues and major organs (brains, lungs, hearts, livers, spleens, kidneys, muscles and intestines) were collected and weighed. The AZD9291 content in these samples was determined by HPLC.
In vivo therapeutic efficacy. The H1975 tumor-bearing mice were established as mentioned above. When the tumor volume reached 100 ~ 200 mm3, the nude mice were randomly divided into 8 groups (n = 5): Control, PFCE NPs (0.1g/kg), AZD9291 (0.5 mg/kg), AZD9291-PFCE NPs (0.5 mg/kg), Control + LIFU, PFCE NPs + LIFU (0.1g/kg), AZD9291 + LIFU (0.5 mg/kg) and AZD9291-PFCE NPs + LIFU (0.5 mg/kg). Free AZD9291 was given via gavage every day and other groups received intravenous injection every 3 days. LIFU was applied to the tumor site after different administrations for 4 h (1.1 MHz, 55.8 mW/cm2, 50% duty cycle, pulse wave mode, 3 W, 20 min). The mice were treated on days 1, 4, 7,10 and 13. The tumor volume and body weight of the nude mice were monitored every other day. Tumor size was measured by T1-weighted MR imaging and vernier caliper. The tumor growth inhibition (TGI) was determined according to tumor weights. The survival rate of mice had been recorded throughout the experiment. All the nude mice were sacrificed on the 14th day after treatment. The tumors of various groups were resected and subjected to Hematoxylin and Eosin (H&E) staining and immunohistochemical staining. The proliferation of tumor cells was assessed by anti-Ki67 antibody (ab15580, Abcam). The microvessel density (MVD) of tumor sections was evaluated by CD31 immuno-histochemical staining. Phosphorylated-EGFR (pEGFR) protein expression status assessed by immunohistochemistry using primary antibodies purchased from Cell Signaling Technology. The sections were observed using an optical microscope. Apoptosis was detected by the TUNEL assay using In Situ Cell Death Detection Kit, according to the manufacturer's instructions and imaged by CLSM.
In vivo biocompatibility studies. H1975 xenograft-bearing nude mice (n = 3) were used in this study. To estimate the biological safety in vivo, H&E staining was carried out on the major organs (heart, liver, spleen, lung, kidney, muscle and intestine) at the end of treatment. Furthermore, mice were intravenously injected with 300 µL of PFCE NPs or AZD9291-PFCE NPs. At different time points (1 and 7 days), blood samples were acquired for electrolytes, blood biochemistry and haematology tests. In addition, to evaluate the biological toxicity of each treatment group, on day 14, the nude mice were killed and blood and serum samples were harvested for hematological examination, biochemical examination of the liver and renal function.
Statistical analysis. Statistical analyses were performed with GraphPad Prism 8.0 software. All values are presented as mean ± standard deviation (SD). One-way or two-way analysis of variance (ANOVA) and the Student’s t test were applied for statistical evaluation, and differences were considered statistically significant at p < 0.05.